Structural basis for methyl-donor–dependent and PNAS PLUS sequence-specific binding to tRNA substrates by knotted TrmD

Takuhiro Itoa,b,c, Isao Masudad, Ken-ichi Yoshidaa,b, Sakurako Goto-Itoa,b, Shun-ichi Sekinea,b,c, Se Won Suhe, Ya-Ming Houd, and Shigeyuki Yokoyamaa,b,f,1

aRIKEN Systems and Structural Biology Center, Tsurumi-ku, Yokohama 230-0045, Japan; bGraduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan; cDivision of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Tsurumi-ku, Yokohama 230-0045, Japan; dDepartment of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107; eDepartment of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea; and fRIKEN Structural Biology Laboratory, Tsurumi-ku, Yokohama 230-0045, Japan

Edited by Joseph D. Puglisi, Stanford University School of Medicine, Stanford, CA, and approved June 19, 2015 (received for review December 10, 2014) The deep trefoil knot architecture is unique to the SpoU and tRNA the N1 position of Ψ1191 in the 18S rRNA (14, 15), but the im- methyltransferase D (TrmD) (SPOUT) family of portance of Nep1 actually resides in its chaperone function, (MTases) in all three domains of life. In , TrmD catalyzes the rather than its methylation function. The TrmD and Nep1 di- N1-methylguanosine (m1G) modification at position 37 in transfer mers share quite similar SPOUT domain structures (Fig. S1B), 36 37 (tRNAs) with the GG sequence, using S-adenosyl-L-methio- but they exhibit largely different intersubunit orientations with 1 nine (AdoMet) as the methyl donor. The m G37-modified tRNA func- distinct elements added to the SPOUT domain (colored blue in tions properly to prevent +1 frameshift errors on the ribosome. Here Fig. S1B). Therefore, it is intriguing to investigate how AdoMet we report the crystal structure of the TrmD homodimer in complex binding may affect substrate RNA binding by TrmD. with a substrate tRNA and an AdoMet analog. Our structural analysis Here, we report the crystal structures of TrmD in complex revealed the mechanism by which TrmD binds the substrate tRNA in with an AdoMet analog, sinefungin (Fig. 1A), in the presence of an AdoMet-dependent manner. The trefoil-knot center, which is struc- wild-type and variant tRNA substrates. These structures revealed BIOCHEMISTRY turally conserved among SPOUT MTases, accommodates the adenosine that the TrmD-specific structural features are the major de- moiety of AdoMet by loosening/retightening of the knot. The TrmD- terminants of tRNA binding and recognition. Furthermore, we specific regions surrounding the trefoil knot recognize the methionine performed structure-guided kinetic analyses of TrmD mutants moiety of AdoMet, and thereby establish the entire TrmD structure for and tRNA variants. Based on these results, we have elucidated global interactions with tRNA and sequential and specific accommoda- tions of G37 and G36, resulting in the synthesis of m1G37-tRNA. the mechanism by which the SPOUT MTase TrmD captures AdoMet in the deep trefoil knot fold and ensures the subsequent 1 36 37 RNA modification | SPOUT methyltransferase | TrmD | X-ray crystallography m G37 methylation of GG -containing tRNAs. Results and Discussion t is highly exceptional for to have a knot in their Structure Determination and Overall Structures. We purified the TrmD Ifolding patterns. Actually, all of the proteins found to possess a proteins from two species, Thermotoga maritima and Haemophilus deep trefoil knot (1–5) belong to a single family of RNA methyl- influenzae, to crystallize in the complex with the T. maritima transferases (MTases), named SPOUT after SpoU (currently called TrmH) and tRNA methyltransferase D (TrmD) (6). SPOUT MTases methylate the base or ribose moiety of ribosomal RNA Significance (rRNA) or transfer RNA (tRNA) in all three domains of life. The deep trefoil knot of the SPOUT MTases provides the binding site In bacterial tRNAs with the 36GG37 sequence, where positions for the methyl donor, S-adenosyl-L-methionine (AdoMet). The 36 and 37 are, respectively, the third letter of the antico- SPOUT MTase transfers the methyl moiety of AdoMet onto its don and 3′ adjacent to the anticodon, the modification of substrate RNA, and produces the methylated RNA and S-adeno- N1-methylguanosine (m1G) at position 37 prevents +1 frame- 1 syl-L-homocysteine (AdoHcy). SPOUT MTases usually function as shifts on the ribosome. The m G37 modification is introduced homodimers, although as an exception, yeast Trm10 functions as by the TrmD, which harbors a deep trefoil knot within a monomer (7). In the homodimeric SPOUT MTases, the helix the S-adenosyl-L-methionine (AdoMet)-binding site. We de- located at the carboxyl terminus of the deep trefoil knot is in- termined the crystal structure of the TrmD homodimer in com- volved in the dimerization (1–5). plex with a substrate tRNA and an AdoMet analog. The structure TrmD, the product of the trmD gene, is one of the broadly revealed how TrmD, upon AdoMet binding in the trefoil knot, conserved SPOUT MTases in bacteria. It is responsible for the obtains the ability to bind the substrate tRNA, and interacts with methylation at the N1 position of G37 in tRNA, in cases where G37 and G36 sequentially to transfer the methyl moiety of 1 the third anticodon letter, 5′ adjacent to G37, is also guanosine, AdoMet to the N position of G37. G36 (Fig. 1A). The modified nucleotide N1-methylguanosine at 1 Author contributions: T.I., S.W.S., Y.-M.H., and S.Y. designed research; T.I., I.M., K.-i.Y., position 37 (m G37) is present in all three domains of life. Re- S.G.-I., S.-i.S., Y.-M.H., and S.Y. performed research; T.I., I.M., K.-i.Y., S.G.-I., S.-i.S., Y.-M.H., markably, mutations in trmD frequently result in growth defects, and S.Y. analyzed data; and T.I., S.W.S., Y.-M.H., and S.Y. wrote the paper. associated with increased +1 frameshift errors (8–12). Each TrmD The authors declare no conflict of interest. monomer consists of two globular domains, the N-terminal do- This article is a PNAS Direct Submission. “ main (NTD), which harbors the SPOUT fold, called the SPOUT Freely available online through the PNAS open access option. ” domain, and the TrmD-specific C-terminal domain (CTD) (Fig. Data deposition: The atomic coordinates and structure factors have been deposited in the S1A) (3, 5). Data Bank, www.pdb.org (PDB ID codes 4YVG, 4YVH, 4YVI, 4YVJ, and 4YVK). So far, only one SPOUT MTase in complex with a substrate RNA 1To whom correspondence should be addressed. Email: [email protected]. • has been crystallized: the Saccharomyces cerevisiae Nep1 RNA This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. complex (Fig. S1B) (13). Nep1 is the SPOUT MTase specific to 1073/pnas.1422981112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1422981112 PNAS Early Edition | 1of9 Downloaded by guest on September 27, 2021 ABacceptor NH2 NH2 sinefungin branch subunit A subunit B CH N N 3 N NH2 N - + - tRNA OOC S OOC N N N N + O + O NH3 NH3

OH OH OH OH AdoMet sinefungin anticodon O O branch H N H3C N N TrmD N G37 G36 H H TrmD H H N N N N N N 90° H sugar H sugar G37 NH2 m1G37 N subunit A N - OOC S N N + O NH3

OH OH AdoHcy

subunit B C Glu80 Glu80

Phe155 Phe155

Glu80 Phe155 Met1 Ser246

Fig. 1. Crystal structure of the TrmD•sinefungin•tRNA complex. (A) Schematic representation of the synthesis of m1G37-tRNA catalyzed by TrmD. The chemical structures of AdoMet, AdoHcy, and sinefungin are also shown. (B) Ribbon representation of the crystal structure of the ternary complex of TrmD, sinefungin, and tRNA. The TrmD subunits A and B are colored green and salmon, respectively. The acceptor and anticodon branches of tRNA are colored brown and yellow, respectively. G36 and G37 in the tRNA are represented by stick models. The sinefungin molecules in the AdoMet-binding sites of the TrmD subunits A and B are represented by CPK models, colored light green and light salmon, respectively. (C) The backbone wire model of the deep trefoil knot structure of subunit A in the tRNA•sinefungin-bound form of TrmD. The backbone model of the residues from Glu80 to Phe155 is presented in a stereoview, from the same angle as the lower panel of B. The color of the residues from Glu80 to Ala153 gradually changes from yellow to blue, to clarify the relationship between the positions in the primary structure and the knotted part in the tertiary structure, as in the bar indicated below. The bound sinefungin is rep- resented by a stick model, and its simulated-annealing omit map, contoured at 3σ, is illustrated on the model.

Gln Gln tRNACUG transcript and sinefungin. T. maritima tRNACUG pos- The crystal structures of the AdoMet-bound and sinefungin- sesses the 36GG37 sequence, which is necessary for recognition bound forms of TrmD are almost the same. The asymmetric unit by TrmD. We successfully crystallized the ternary complex of contains one TrmD subunit bound to one AdoMet or sinefungin Gln molecule, and a homodimer is formed between the symmetry- H. influenzae TrmD, the T. maritima tRNACUG transcript, and sinefungin. Actually, the structure of H. influenzae TrmD was related subunits (Figs. S1A, S3B,andS4A). H. influenzae TrmD – – previously well characterized (3), and became the basis for the consists of the NTD (residues 1 160) and the CTD (residues 169 – further structural characterization. Although the enzyme and 246) (Fig. S1A), and the interdomain linker of residues 161 168 is the substrate are from different species, the T. maritima disordered in the tRNA-free structures. These characteristics are Gln consistent with those previously reported for the AdoMet- and tRNACUG transcript is methylated by H. influenzae TrmD 2.2- to 99-fold more efficiently than the H. influenzae tRNA tran- AdoHcy-bound TrmD structures (PDB ID codes 1UAK, 1UAL, 1UAM, and 1P9P) (3, 5). Minor conformational differences were scripts (Fig. S2) (rows A, E, F, and G in Table 1). Therefore, identified in the sugars and between the methionine and methi- the interaction between H. influenzae TrmD and the T. maritima onine-mimicking moieties of AdoMet and sinefungin (Fig. S4A). tRNAGln transcript is sufficiently functional. We determined the CUG The sugar moieties of AdoMet and sinefungin adopt the C2′-and structure of this ternary complex of H. influenzae TrmD, sinefungin, C3′-endo conformations, respectively, probably because of the • andtRNA,referredtoasthetRNA sinefungin-bound form here- chemical differences between the methyl donor and the analog. after, at 3.0 Å resolution (Fig. 1B; see also Fig. S3A). We also de- In the crystal structure of the tRNA•sinefungin-bound form, termined high-quality structures of the tRNA-free H. influenzae the asymmetric unit contains the TrmD homodimer in complex TrmD•AdoMet and TrmD•sinefungin binary complexes, referred with one tRNA molecule (Fig. 1B and Fig. S3A). However, each to as the AdoMet-bound and sinefungin-bound forms hereafter, at of the two catalytic pockets holds a sinefungin molecule, similar 1.55 Å and 1.6 Å resolutions, respectively (Figs. S1A, S3B,and to the sinefungin-bound form, in which sinefungin is partially S4A). The data collection and refinement statistics are summarized buried in the cavity constructed by the deep trefoil knot structure in Table S1. (Fig. 1C). The substrate tRNA interacts with the catalytic pocket

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1422981112 Ito et al. Downloaded by guest on September 27, 2021 Table 1. Steady-state kinetics of H. influenzae TrmD PNAS PLUS −1 −1 −1 Row Enzyme tRNA substrate kcat (s ) Km (μM) kcat/Km (s ·μM ) Relative

A Hin TrmD WT Tma tRNAGln WT 0.24 ± 0.01 0.82 ± 0.15 0.29 1 B Hin TrmD WT Tma tRNAGln G36A 0.36 ± 0.06 2.7 ± 0.9 0.13 1/2.1 C Hin TrmD WT Tma tRNAGln G36C 0.36 ± 0.03 184 ± 16 0.002 1/150 D Hin TrmD WT Tma tRNAGln G36U 0.17 ± 0.02 114 ± 1 0.0015 1/190 E Hin TrmD WT Hin tRNAPro 0.13 ± 0.01 1.0 ± 0.1 0.13 1/2.2 F Hin TrmD WT Hin tRNALeu 0.32 ± 0.01 8.0 ± 1.1 0.04 1/7.1 G Hin TrmD WT Hin tRNAArg 0.078 ± 0.003 27 ± 1 0.0029 1/99 H Hin TrmD Arg154Ala Tma tRNAGln WT 0.0014 ± 0.0002 64 ± 12.2× 10−5 1/13,000 I Hin TrmD Ser165Ala Tma tRNAGln WT 0.34 ± 0.03 4.4 ± 0.3 0.078 1/3.7 − J Hin TrmD Asp169Ala Tma tRNAGln WT 0.0047 ± 0.0001 67 ± 19 7.0 × 10 5 1/4,100

Hin, Haemophilus influenzae; Tma, Thermotoga maritima; WT, wild-type.

involving the knot of subunit A, the NTDs of subunits A and B, negative charge on O6 of G37 is stabilized by the positively and the neighboring CTD of subunit B (Fig. 1B). The identified charged guanidine group of the strictly conserved Arg154*. In a + stoichiometry between the TrmD monomer, sinefungin, and recent study, a Mg2 ion was found to stabilize the developing tRNA of 2:2:1 is consistent with the previous biochemical results negative charge on O6 of G37 after proton abstraction, thus (16). Upon tRNA binding, the CTD of subunit B changes its contributing to the catalytic mechanism of methyl transfer (19). conformation to snugly contact the tRNA (arrow in Fig. S4B), In the mFo-DFc map of the tRNA•sinefungin-bound form, a and therefore the TrmD dimer is no longer conformationally weak electron density was identified near N7 of G37, which might + symmetric. Consistent with biochemical studies (17), TrmD re- be a partially bound Mg2 ion (Fig. S6B). The deprotonated N1 cognizes only the tRNA anticodon branch, composed of the D atom of G37 then nucleophilically attacks the methyl group of and anticodon arms, and the variable region (colored yellow in AdoMet, generating the m1G37-modified tRNA with AdoHcy as Fig. 1B), and lacks any contacts with the acceptor branch, formed the leaving group. by the acceptor and T arms (colored brown in Fig. 1B). The BIOCHEMISTRY interdomain linker of subunit B forms a helix upon tRNA The Interaction with G36. As shown in Fig. 2B, the 1-NH and 2-NH2 binding, and participates in the interaction with the substrate groups of G36 interact with the side-chain carboxyl group of tRNA (Fig. 1B and the red dotted circle in Fig. S4B). Asp50, which is strictly conserved in TrmD (Fig. S5A). This interaction stabilizes the guanine base in the syn conformation, The Interaction with G37. In the tRNA•sinefungin-bound form, which is rarely observed in tRNA molecules (Fig. 2B). The the base moiety of G37 is flipped out from the anticodon loop, guanine base at position 36 is sandwiched between the bases of and protrudes into the catalytic pocket (Figs. 1B and 2A). The 1-NH and 2-NH2 groups of G37 hydrogen bond with the side- chain carboxyl group of Asp169 of subunit B. Hereafter, a resi- “ ” A due name with an asterisk, such as Asp169*, indicates that of Ser165 Ser165 subunit B, and a residue name without an asterisk refers to that of subunit A. In all TrmD , this position is occupied by a negatively charged residue, such as aspartate or glutamate (Fig. Leu160 Leu160 S5). The side-chain of Leu160* stacks onto the guanine base, and the γ-OH group of Ser165* hydrogen bonds with the 2′-OH group of G37. In addition, the side-chain of Arg154* is close to the G37 G37 guanine base. The e amino group of sinefungin, corresponding to Asp169 Asp169 the donor methyl group of AdoMet, is located next to the 1-NH sinefungin sinefungin group of G37, which is a suitable position for methyl-transfer after Arg154 Arg154 deprotonation of the 1-NH proton. The identified G37-interaction manner (Fig. 2A) is consistent with the results of a study of tRNAs containing guanosine analogs of G37 (18). B Next, we performed kinetic analyses with TrmD mutants, to evaluate the importance of the aforementioned residues (Table 1). 83A 83A The Arg154Ala and Asp169Ala mutations essentially abolished Asp50 Asp50 the activities. These mutations affected both the k and K and cat m 63G 63G decreased the kcat/Km values of Arg154Ala and Asp169Ala by 13,000- and 4,100-fold, respectively (rows H and J in Table 1). Therefore, these two residues play important roles in the catalysis 53U 53U of methyl transfer, consistent with the previous biochemical studies of Escherichia coli TrmD (5). In contrast, the Ser165Ala mutation had a modest influence on the TrmD activity (compare row I with row A in Table 1), consistent with the data showing that the 2′-OH group of G37 is not critical for TrmD (18). Based on our crystal structure and kinetic analyses, we pro- Fig. 2. TrmD interactions with G37 and G36 in the target tRNA. (A) Close-up views of the G37-binding pocket (stereoview). The molecules are colored as in pose a mechanism for methyl transfer by TrmD (Fig. S6A). First, Fig. 1B, except that sinefungin is colored gray. The interacting residues of the negatively charged side-chain carboxyl group of Asp169* func- TrmD and sinefungin are indicated by stick models. The hydrogen bonds be- tions as a catalytic general base, and abstracts the proton from tween TrmD and tRNA are indicated by gray dotted lines. (B) Close-up views of 1 N of G37. In the resultant intermediate state, the developing the G36-binding pocket (stereoview). The molecules are depicted as in A.

Ito et al. PNAS Early Edition | 3of9 Downloaded by guest on September 27, 2021 A38 and U35. The distance between the N9 atoms of G36 and Comparison of the Structures in the Absence and Presence of the G36 A38 is only 4.1 Å, which is shorter than the corresponding dis- Interaction. To reveal how the local G36 interaction influences tance between the adjacent stacking residues in a regular RNA the overall substrate interaction and the enzymatic reaction, we • double helix (∼4.8 Å), and there is no space for G37 to be successfully determined the crystal structures of TrmD sinefungin inserted between the bases of G36 and A38. Thus, the identified in complex with the G36U or G36C tRNA variant (Table S1). In G36-interacting pocket is formed only after the guanine base at each complex, the stoichiometry of the tRNA to the enzyme dimer remained the same as that in the wild-type complex. Because the position 37 is flipped from the loop and held by TrmD, indicating tertiary structures with these two variants are almost the same, that the G36 interaction occurs after the G37 interaction. hereafter the structure of the G36U tRNA variant (Fig. 3A)is To examine the influence of the G36 interaction on the methyl used for comparison with the wild-type structure (Fig. 3B). transfer reaction, we analyzed TrmD kinetics with the tRNA As expected from our kinetic experiments, we observed that variants G36A, G36U, and G36C, as well as the wild-type. The the uracil base at position 36 does not interact with Asp50 in the G36A variant was methylated by TrmD almost as efficiently as variant structure, and is directed to the outside of the anticodon the wild-type, although the Km value was slightly higher than that loop with the regular anticonformation (Fig. 3A). Because the of the wild-type (rows A and B in Table 1). The 6-NH2 group of G36 interaction with Asp50 is lost in the U36-tRNA structure, G36A is probably recognized by one of the carboxyl oxygen the extensive stacking between A38:C32 and G36 is also absent atoms of Asp50. It is noteworthy that the 36AG37 tRNA sequence (Fig. 3A). In addition, the hydrogen-bonding partner of the does not naturally occur in bacteria. In contrast, the methyl main-chain NH group of the strictly conserved Gly59 is different: 1P 2P transfer efficiencies with the G36C and G36U variants fell to 1/150 it is the O atom of A38 in the variant structure but the O and 1/190, respectively, of the wild-type level (rows A, C, and D in atom in the wild-type structure, as indicated by the dotted lines Table 1). The K values of the G36C and G36U variants were in Fig. 3. Thus, we have designated these anticodon-arm struc- m “ ” “ ” more than 100-times higher than that of the wild-type, whereas the tures as the loose and tight forms in the absence and pres- k values changed only slightly. Presumably, the side-chain of ence, respectively, of the G36 interaction. cat Although many differences have been identified between the Asp50 is too short to capture a pyrimidine base. We measured the wild-type and variant structures, the coordinates of the G37 base affinities between sinefungin-bound TrmD and tRNA variants, as are quite similar between them (Fig. 3). This observation strongly well as the wild-type, by monitoring the quenching of the intrinsic suggests that the interaction of TrmD with G37 is independent of tryptophan fluorescence of TrmD (Fig. S7). Compared with the the interaction with G36, which is consistent with the hypothesis dissociation constants for the wild-type tRNA (0.28 μM) and the that the G36 interaction is established only after the G37 base is G36A tRNA variant (0.41 μM), TrmD exhibited largely reduced flipped out from the anticodon loop. affinities for the G36C and G36U tRNA variants (Kdswerenot Regarding TrmD in the variant structure, the orientation of determined), consistent with the Km values described above. the His46 side-chain is quite different. The imidazole ring flips

A Gly59

Asp50 Ser165 A38 C32 His46 sinefungin U36 or C36 Lys162 G37 U36

U35 G36U 30 150 B Gly59 Asp50 C32 Lys162 A38 Ser165 sinefungin G36 G37 G36 U35 His46

WT 50 150

Fig. 3. The structural changes induced by the G36 interaction. (A) Ribbon representation of the ternary complex of TrmD, sinefungin, and the G36U variant tRNA (Left), together with the schematic representation (Right Upper), and the stick model colored with B-factors (Right Lower). The key residues are depicted by stick models and labeled. The between Gly59 and Pro58 is depicted by a stick model, and the hydrogen bond from the NH group of Gly59 is indicated by the gray dotted line. The viewpoint of the left panel is indicated by the black arrow in the schematic representation. The red dotted square corresponds to the region represented in the B-factor-colored model in the right lower panel. The colors of the molecules are fainter than thoseofthe wild-type, as in Fig. 2. (B) Representations of the ternary complex of TrmD, sinefungin, and the wild-type tRNA, in the same manner as in A.

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1422981112 Ito et al. Downloaded by guest on September 27, 2021 outside in the variant structure, while it stacks on the C32 base in tRNA is likely to be established by the recognition of the phos- PNAS PLUS the wild-type structure (Fig. 3), where the interaction between phate groups on the 5′ strand of the anticodon arm, by the CTD of His46 and C32 would contribute to the conformational change of subunit B and the NTD of subunit A (Fig. 4B). The main-chain the anticodon arm. Most importantly, the interdomain helix NH group of Gly55 and the side-chains of Arg52, Arg183*, and between the NTD and the CTD, which becomes helically or- Tyr54, which are highly conserved in TrmDs (Fig. S5A), form dered upon wild-type tRNA binding, remains disordered in the networks with the phosphate groups of G26, G27, and C28, by variant structure (Fig. 3A), presumably because of steric hin- electrostatic and hydrogen-bonding interactions. Finally, the drance between the interdomain helix and the anticodon arm of main-chain NH group of Gly59 interacts with the phosphate the variant tRNA. If the helix was formed in the variant struc- group of A38, located 3′ adjacent to the target position 37. This ture, then the side-chain of Lys162* in TrmD would collide with cascade of interactions allows TrmD to find and recognize the the sugar moiety of A38 in the tRNA (Fig. 3). The interdomain base moiety at position 37 in the tRNA. region of the variant seems to be quite mobile, as indicated by its higher B-factors (Fig. 3A), and waters may easily enter the G37- Structural Changes of TrmD upon AdoMet Accommodation. A com- interacting pocket, which would weaken the hydrogen bonds parative analysis of the tRNA•sinefungin-bound, AdoMet-bound, between Asp169* and G37. However, the stable formation of the and previously determined apo (PDB ID code 1UAJ) (3) TrmD interdomain helix prevents waters from entering the G37-inter- structures revealed several key differences around the AdoMet- acting pocket (Fig. 3B). The disordered state of the interdomain binding site. The most notable is the orientation of Phe171* in the linker was also observed in the tRNA-free binary complexes. tRNA•sinefungin-bound form (Fig. 5A and Fig. S8A), which is Based on the ordering of the interdomain linker upon tRNA inserted between the CTD of subunit B and the NTD of subunit A. binding, we suggest that the insertion of G37 into the Phe171* is next to the residues that form a pocket for the carboxyl enables the interaction between Asp50 and G36, which induces and amino groups of the methionine-mimicking moiety of sine- additional conformational changes that further strengthen the fungin. Other residues of the pocket include Gln90, Ser170*, and interactions with G37 for the methyl transfer reaction. Asp177* (Fig. 5A). This orientation of Phe171* was also observed in the AdoMet-bound form, even though the interdomain linker Selection of tRNA Anticodon Branch and Detection of Position 37. was disordered. Intriguingly, the specific orientation of Phe171* The manner in which TrmD interacts with the D and anticodon was not observed in our previously determined TrmD structure in stems determines how TrmD searches for position 37 in sub- complex with adenosine, lacking the methionine moiety (PDB ID

strate tRNAs. As shown in Fig. 4A, the CTD of subunit B binds code 3AXZ) (Fig. S8B) (20). It appears that the interaction be- BIOCHEMISTRY to the D-stem end. The conserved Ser-Gly-His/Asp-His residues, tween the methionine moiety of AdoMet and the binding pocket located between helices α7 and α8 in the CTD, recognize the of TrmD is necessary for the stable insertion of Phe171*. minor groove next to the G10:C25 base pair, which is conserved In the apo form, in addition to the disorder of the interdomain in all of the substrate tRNAs in H. influenzae. The identified linker, the following five residues from Gly161* to Asp173* in recognition manner indicates that TrmD is able to similarly the CTD are also disordered, probably because of the lack of recognize the G10:U25 pair in the TrmD•substrate tRNAs from interaction with any ligand, such as AdoMet (Fig. S8A) (3). In other species. However, the strong binding between TrmD and the apo form, Phe171* is not inserted as in the AdoMet- or tRNA•sinefungin-bound form; instead, the side-chain of Leu223* of the CTD is inserted into the corresponding groove, whereas the CTD is displaced, especially around the α9helix(Fig. S8A). A How does TrmD accommodate the AdoMet molecule? In the AdoMet-bound form, the adenosine moiety of AdoMet, espe- cially the adenine-base part, is buried in the deep trefoil knot His200 His200 structure of TrmD (state IV in Fig. 5B). However, the structure 01G 01G of the deep trefoil knot fold does not seem to be capable of Ser198 Gly199 Ser198 Gly199 accommodating AdoMet within its binding site in TrmD without any steric hindrance (state I in Fig. 5B). These considerations 52C 52C raised the possibility that TrmD undergoes a conformational change during AdoMet accommodation. 102siH 102siH We therefore investigated the TrmD movement that would allow the accommodation of AdoMet. First, we looked at the B-factors around the AdoMet-binding sites of the determined structures, and found that the 88SPQG91 loop (referred to as the “cover loop,” B hereafter) in the middle of the deep trefoil knot structure (colored Gly55 72G 62G Gly55 72G 62G magenta in Fig. 5B), and the side-chains of Arg215* and Arg219* in 65ylG 65ylG the CTD, have relatively high B-factors in the apo form (Fig. S9A). 82C 82C Gly57 Gly57 Manual modeling of the structure revealed that the small movements Pro58 45ryT Pro58 45ryT of the side-chains of Arg215* and Arg219* create space for the 381grA 381grA 88SPQG91 cover loop to open the AdoMet-binding site. The cover- loop motion may involve the movement of the entire CTD, because Pro53 Arg52 Pro53 Arg52 95ylG 95ylG of its relatively high B-factors in both the apo and AdoMet-bound 83A 83A forms (Fig. S9 B and C). Because the knot structure is not involved in the crystal packing contacts (Fig. S3), the B-factors around the knot structure are affected mainly by the mobility of the structure. As a result, the open conformation of the 88SPQG91 cover loop allows Fig. 4. The interaction between the anticodon branch and TrmD to find po- B sition 37. (A) The loop between helices α7andα8 recognizes the minor groove AdoMet to enter its binding site (from state II to state III in Fig. 5 ). next to the G10:C25 pair (stereoview). The key residues in TrmD are depicted by In other words, the deep trefoil knot of TrmD can be loosened, thus stick models. The identified electrostatic and hydrogen bonds are indicated by opening the pocket to accommodate AdoMet. After accommodating the dotted gray lines. (B) The recognition of the phosphate groups of G26, G27, the adenine base moiety, the knot can be retightened, which results in C28,andA38byTrmDisindicatedinthesamemannerasinA. the AdoMet-bound form (state IV in Fig. 5B)(3).

Ito et al. PNAS Early Edition | 5of9 Downloaded by guest on September 27, 2021 A tRNA•sinefungin-bound tRNA•sinefungin-bound

322ueL 322ueL

171ehP 171ehP 071reS 071reS 09nlG 09nlG 771psA 771psA

73G 73G sinefungin sinefungin

B AdoMet-binding stage

SPQG cover loop AdoMet AdoMet-binding I site II III IV

“Tight-knot” state “Loose-knot” state Adenosine-moiety Adenosine-moiety AdoHcy recognition recognition V (loose-knot state) (tight-knot state)

XI Methionine-moiety Phe171 Methyltransfer stage recognition loop

Methyltransfer reaction m1G37-tRNA Methionine-moiety recognition tRNA X IX VIII VII VI

“tight-form” “loose-form” tRNA tRNA Interdomain-helix G36 recognition G37 recognition Stem recognition Stem recognition formation (step 0) (step −1) (step −2) tRNA-binding stage

Fig. 5. A model of the enzymatic cycle of TrmD. (A) The AdoMet-binding site of the tRNA•sinefungin-bound TrmD (stereoview). The residues recognizing the methionine-mimic moiety of sinefungin are depicted by stick models, and the identified hydrogen bonds are indicated by the dotted gray lines. The model lacks the NTD of subunit B to facilitate visualization, and is colored as in Fig. 2, except that the 88SPQG91 cover loop is colored magenta. (B) Based on the crystal structures, the kinetic experiments and the structural analyses, the recognition order of AdoMet and the elements in the 36GG37-containing tRNA, and the subsequent methyl transfer reaction by TrmD are presented. The states in the AdoMet-binding stage (states I to V) are illustrated as close-up viewsofthe AdoMet-binding site with schematic trefoil-knot structures, those in the tRNA-binding stage (states VI to X) are illustrated around the tRNA-binding site, and the methyltransfer stage (state XI) is illustrated schematically. The TrmD, AdoMet, and tRNA in the graphical panels are depicted by surface models, CPK models, and stick models with ribbons, respectively. The figures are colored as in Fig. 2, except that the nucleotides at positions 36 and 37 are colored cyan, the 88SPQG91 cover loop is colored magenta, and the ordered loop in the CTD and the interdomain helix are colored purple. The right panel of state I, the panels of states II and III, and the left panel of state IV lack the CTD of subunit B, to facilitate visualization.

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1422981112 Ito et al. Downloaded by guest on September 27, 2021 We then examined the AdoMet-binding sites of other SPOUT inward movement of the A38 sugar moiety provides space for the PNAS PLUS MTases. The AdoMet-binding site of the SPOUT domain mainly TrmDinterdomainlooptofoldintoahelixjustaboveG37(stateX). consists of three loops from the deep trefoil knot: the cover, wall, TrmD traps the substrate tRNA tightly at this moment and is and bottom loops (colored magenta, light blue, and orange, re- ready for the reaction. The methyl moiety of AdoMet is trans- spectively, in Figs. S5B and S9D). The bottom loop, corresponding ferred to the N1 atom of G37 (state XI). After methyl transfer, to residues Ser132 to Gly140 in TrmD, constructs the bottom of the the m1G37-modified tRNA and the other product, AdoHcy, are AdoMet-binding site, and touches the other dimerizing SPOUT released from TrmD, accompanied by the collapse of the inter- domain. The wall loop corresponds to residues Gly113 to Ile118 in domain helix and the loosening of the knot structure. Finally, the TrmD, and forms a wall next to the methionine moiety of AdoMet. resultant apo-form TrmD heads to the next round of the re- The cover loop corresponds to the residues 88SPQG91 in TrmD, and action. To facilitate the visualization of this TrmD enzymatic buries the adenosine moiety, as described above. This architecture mechanism, a movie file is available (Movie S1). Gln of the three loops is common among six of the structurally char- The tRNACUG regions in direct contact with TrmD are strictly acterized SPOUT domains (Fig. S9E). In all cases, the B-factors of conserved in other TrmD substrate tRNAs, including the three the cover loop are higher than those of the wall and bottom loops in H. influenzae tRNA transcripts analyzed in this study. Therefore, the overall enzymatic cycle is likely to be generally conserved. However, both the AdoMet(-analog)/AdoHcy-bound and apo forms (Fig. Gln S9F). Therefore, the mechanism for AdoMet-accommodation by as described above, TrmD methylates the T. maritima tRNACUG TrmD, starting with the opening of the cover loop, seems to be transcript 2.2- to 99-fold more efficiently than the H. influenzae common among the SPOUT MTase family members (Fig. S9D). tRNA transcripts (rows A, E, F, and G in Table 1). Consequently, the tRNA sequences outside the direct contact regions—for exam- The Overall Enzymatic Cycle of TrmD. Based on the results described ple, the D and T arms—might influence the properties of the direct above, we propose a model for the TrmD enzymatic cycle, which contact regions, and thus indirectly affect the reactivity of TrmD “ ” is divided into three major stages: the AdoMet-binding, tRNA- ( indirect readout of the characteristics of tRNA substrates). binding, and methyl transfer stages (Fig. 5B). “ Why Do Bacteria Keep the 36GG37 Sequence in tRNAs? It would seem The apo-form TrmD is likely to shuttle between the tight- 36 37 knot” (state I) and “loose-knot” (state II) states, by establishing easier for bacteria to replace the GG sequence in tRNANNG by the 36GA37 sequence, for preventing +1 frameshift errors. The an equilibrium between the closed and open conformations of the 36 37 “ ” fact that bacteria keep the GG sequence together with TrmD cover loop, and only TrmD in the loose-knot stateisableto 1 1 to synthesize m G37 leads to the hypothesis that the N -methylation BIOCHEMISTRY accommodate AdoMet without any steric hindrance (state III). In 36 37 the loose-knot state, the adenosine moiety of AdoMet is captured status of G37 of the GG -containing tRNAs functions to control gene expression. One interesting example in bacteria is that the +1 in the hollow constructed by the deep trefoil knot structure in frameshift is used for the autogenous regulation of the translation of TrmD (state IV), whereas the methionine moiety is recognized by release factor 2 (RF-2), which functions to terminate translation at Gln90, Ser170*, and Asp177* (stateV).TheAdoMetrecognition the UGA and UAA codons (21, 22). The early region of the RF-2 induces the stable insertion of the Phe171* side-chain, allowing the mRNA contains the in-frame UGA termination codon, as follows: CTD to assume the tRNA-binding form preferentially and tran- Leu UAU CUU UGAC UAC. When the RF-2 level is low, a tRNA sitioning the structure into the tight-knot state. The importance of with 36GG37 and lacking the m1G37 modification can shift into the the insertion of Phe171* is consistent with the fact that the Ala +1 frame with the UUU codon, rather than the in-frame CUU substitution in E. coli TrmD abolishes the enzyme activity (5). codon at the P-site of the ribosome. This frameshift results in the The AdoMet-bound TrmD then searches for a substrate tRNA. translation of the GAC codon in the +1 frame to asparagine, as the We hypothesize that TrmD binds first to the anticodon stem of the residue next to the leucine. A lower level of RF-2 may be present substrate tRNA with the canonical shape of the anticodon loop, and because of slower bacterial growth and limiting metabolites (23, 24). then the loop conformation changes to insert G37 into its binding The limiting metabolites include ATP and methionine, which would pocket within TrmD (state VIII). However, if the tRNA anticodon lead to reduced AdoMet levels and decreased synthesis of m1G37- stem binds in the manner observed in the crystal structures, then the tRNA by TrmD. Thereby, the lower levels of m1G37 would induce canonical anticodon loop conformation sterically clashes with TrmD. higher frequencies of +1 frameshifting of the tRNALeu,fortrans- In fact, TrmD interacts mainly with the phosphate groups at posi- lation of the full-length RF-2 protein. In other words, bacterial cells tions 26, 27, and 28 of the anticodon stem, from the minor groove may use the synthesis of m1G37-tRNA to control the level of the +1 “ ” side, in the crystal structures ( step 0 ). We therefore hypothesized frameshift, and thus regulate gene expression. that TrmD can interact with the phosphate groups at positions 27, Although TrmD synthesizes m1G37-tRNA in bacteria, a differ- 28, and 29 (“step –1”) or positions 28, 29, and 30 (“step –2”). The ent enzyme, Trm5, performs the same enzymatic function in eu- steric hindrance with the canonical conformation of the anticodon karyotes and (25–28). The lack of homology between loop is much less in step –1thaninstep0,andisnegligibleinstep–2. TrmD and Trm5—and the broad conservation of TrmD among Therefore, step –2 was postulated to be the first step of the anti- bacterial species—have led to the focus on TrmD as a leading codon binding (state VI). Then, TrmD and the anticodon stem are target for the next generation of antibiotics worthy of priority likely to mutually slide to step –1, together with the conformational consideration (29). Potent inhibitors of TrmD, which are strong change of the inherently flexible anticodon loop (state VII). Finally, candidates for new antibiotic drugs, were recently reported (30). onemoreslidetostep0allowsthe main-chain NH group of Gly59 The present structural study provides the basis for designing robust to capture the phosphate group at position 38, which becomes a and selective inhibitors of TrmD as a novel antimicrobial strategy. landmark for the insertion of the base moiety at position 37 within the catalytic pocket (state VIII). Once the base at position 37 is Materials and Methods judged as a guanine and interacting tightly, the pocket for position Preparation of Proteins and tRNA Transcripts. The ORF of H. influenzae TrmD 36 searches for G36 (state IX). Immediately after G37 recognition, was cloned into the NdeI/XhoI sites of pET-28b(+), and the expressed protein the anticodon arm is probably in the “loose form,” as in the crystal has a His6-tag at the N terminus. E. coli strain Rosetta2 (DE3) was transformed by structures with U or C at position 36. The guanine base in the loose the recombinant plasmid and was cultured in LB medium. Protein synthesis was induced by adding isopropyl β-D-1-thiogalactopyranoside to a final concentration form at position 36 can flip to the syn conformation. With the rec- of 0.5 mM. Cells were cultured at 303 K for 5 h after induction and were har- ognition of the syn-form guanosine at position 36, the structures vested. The cells were lysed by sonication, and the debris was removed by cen- of the TrmD loops around G36 change, and the tRNA anticodon trifugation. The cleared lysate was purified by affinity chromatography using Ni arm also undergoes a structural alteration to the “tight form.” The Sepharose 6 Fast Flow (GE Healthcare), by ion-exchange column chromatography

Ito et al. PNAS Early Edition | 7of9 Downloaded by guest on September 27, 2021 using RESOURCE Q or Mono Q (GE Healthcare), and by gel-filtration column change for the first strong binding, E is the total concentration of the TrmD chromatography using Superdex 200 (GE Healthcare). The purified TrmD dimer, A is the slope (the rate of the fluorescence signal change with respect protein was stored at 277 K in 50 mM Tris·HCl buffer (pH 8.0), containing 100 mM to the tRNA concentration) for the weak binding, and T is the total con- NaCl and 5 mM 2-mercaptoethanol. centration of tRNA. The data were fit to the above equations, using the The site-directed mutations of the trmD gene were introduced using a KaleidaGraph software. PrimeSTAR Mutagenesis Basal Kit (Takara), and the mutants were purified in the same manner as wild-type TrmD. Crystallization, Data Collection, and Refinement. For crystallizations of the All tRNA transcripts were prepared by in vitro transcription using T7 RNA TrmD•AdoMet and TrmD•sinefungin binary complexes, TrmD was concen- polymerase, and the transcribed tRNAs were purified by RESOURCE Q ion- trated to about 10 mg/mL, using an Amicon Ultra filter (Millipore). A 1.2-μL exchange chromatography (GE Healthcare). The purified and desalted tRNA portion of the protein solution, supplemented with 1 mM AdoMet or sine- · transcripts were dissolved in 20 mM Tris HCl (pH 7.6) buffer containing 5 mM fungin, was mixed with an equal volume of the reservoir solution, consisting MgCl2, heated at 353 K for 3 min, and then gradually cooled to room of 0.8–0.85 M sodium citrate and 0.1 M N-cyclohexyl-2-aminoethanesulfonic temperature for annealing. acid (pH 8.0–8.4). A crystallization drop was formed on an MRC-2 crystalli- zation plate (Swissci) containing a 70-μL reservoir solution in the well, and Methylation Kinetics Analysis. Steady-state kinetic analyses were performed was incubated at 293 K. Within a few days, crystals appeared in the drop. according to the procedure described previously (31). Each tRNA sample (final The crystals were flash-cooled with liquid nitrogen in a cryoprotectant re- μ concentration ranging from 0.1 to 80.0 M, depending on the combination of agent containing 25% (vol/vol) polyethylene glycol (PEG)-400. the enzyme and the substrate tRNA) was denatured by heating at 85 °C for For crystallization of the TrmD•tRNA•sinefungin ternary complex, equal 3 min and annealed at 37 °C before use. The tRNA was mixed with a saturating volumes of the protein and tRNA solutions, at a molar ratio of 5:1, were amountof[3H-methyl]AdoMet (30 μM) in the reaction buffer, containing 0.1 M mixed. This solution was concentrated using an Amicon Ultra filter (Milli- Tris·HCl, pH 8.0, 4 mM DTT, 0.1 mM EDTA, 6 mM MgCl ,24mMNHCl, and 2 4 pore) to a final total concentration of about 5 mg/mL. A 1.2-μL portion of 0.024 mg/mL BSA, and then mixed with the H. influenzae TrmD enzyme (final the sample solution, supplemented with 1 mM sinefungin, was mixed with concentration ranging from 0.02 to 5.0 μM). Aliquots were taken at various 1.5-μL reservoir solution, containing 0.1 M sodium acetate trihydrate time points and precipitated by 5% (wt/vol) trichloroacetic acid on filter pads, (pH 4.6), 0.8 M ammonium phosphate monobasic, and 4% (wt/vol) PEG- and the amount of methyl transfer was determined by quantification of [3H] 20,000. The same crystallization plate as in the case of the binary complex incorporation, using a scintillation counter. After correction of the quenching was used and crystals appeared within a few days. The crystals were flash- factor from filter precipitation, the initial rate at each tRNA concentration cooled with liquid nitrogen in a cryoprotectant reagent containing 30% (pmole/min) was calculated and plotted as a function of the concentration. (vol/vol) ethylene glycol. The TrmD crystals in the complexes with the G37U The data were fit to the Michaelis–Menten equation using the KaleidaGraph or G37C tRNA variants were prepared by a similar method to that for the software (Synergy Software), and the K , k ,andk /K values for each m cat cat m wild-type tRNA. tRNA and enzyme were calculated from the curve fitting. X-ray diffraction datasets were collected on beamlines at the Photon Factory (Ibaraki, Japan) and at SPring-8 (Hyogo, Japan), as described in Table Fluorescence Analysis of tRNA-Binding. The interactions between sinefungin- bound TrmD and tRNAs were monitored by measuring the quenching of S1. The datasets were processed with either the HKL-2000 software (32) or intrinsic tryptophan fluorescence. The fluorescence was excited at 295 nm, the XDS software (33). The initial phases were determined by the molecu- and the emission was monitored at 310–400 nm at room temperature in lar-replacement method, using the program Phaser (34). The crystal struc- fluorescence measurement buffer [20 mM Tris·HCl buffer (pH 8.0), contain- ture of the apo-form of H. influenzae TrmD (PDB ID code 1UAJ) (3) was • ing 50 mM NaCl, 5 mM MgCl , 2.5 mM 2-mercaptoethanol, and 10 μM used as the search model for the structures of the TrmD AdoMet and 2 • sinefungin]. The TrmD dimer and tRNA variants were mixed to achieve final TrmD sinefungin binary complexes. For solving the initial phase of the Gln concentrations of 0.2 μM and 0–10 μM, respectively, and 750-μL samples ternary complex with the wild-type T. maritima tRNACUG, the tertiary struc- • were measured using a Spectrofluorometer FP-8500 (Jasco). The fluores- tures of the currently determined TrmD sinefungin binary complex and the • cence intensities at 330 nm were used for the fittings. To obtain the disso- tRNA portion of the GluRS tRNA complex (PDB ID code 3AKZ) (35) were used. The initial phases of the complex structures with the tRNA variants were ciation constants for the first strong binding (Kd1) with the wild-type and G36A tRNAs, the quadratic equation was used for the fitting. The data did calculated, using the coordinates of the currently determined ternary com- not reach a plateau within the tRNA concentration range. Additionally, the plex with the wild-type tRNA. The models were refined using the programs dissociation constants for the second binding with the wild-type and G36A Coot (36), Refmac (37), CNS (38, 39), and Phenix (40). The dataset and re- tRNAs and those for the first and second bindings with the G36C and G36U finement statistics, together with the accession codes, are tRNAs appeared unusually high. For these reasons, only the linear fits were provided in Table S1. The molecular graphics were prepared with the pro- applied, and the dissociation constants were not determined. Therefore, the gram PyMol (Schrödinger). equation for the wild-type and G36A tRNAs was, n o = ACKNOWLEDGMENTS. We thank the staffs of the beamlines BL32XU and 2 1 2 F = F0 − ΔF1 × ½ E + T + Kd1 − E + T + Kd1 − 4 × E × T = 2 × E + A × T BL41XU at the SPring-8 and the beamlines BL-5A, AR-NW12A, and AR-NE3A at the Photon Factory for their support during data collection. This work was [1] performed under the approval of the Japan Synchrotron Radiation Research Institute (Proposal 2011A1217), and that of the Photon Factory Program and that for the G36C and G36U tRNAs was, Advisory Committee (Proposal 2011G547). This work was supported by the F = F0 + A × T, [2] Targeted Proteins Research Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan, JSPS KAKENHI Grant where F is the measured fluorescence signal intensity, F0 is the fluorescence 20247008 (to T.I. and S.Y.), and National Institutes of Health Grant GM081601 signal intensity without the tRNAs, ΔF1 is the scale of the fluorescence signal (to Y.-M.H.).

1. Michel G, et al. (2002) The structure of the RlmB 23S rRNA methyltransferase reveals a 8. Byström AS, Björk GR (1982) Chromosomal location and cloning of the gene (trmD) new methyltransferase fold with a unique knot. Structure 10(10):1303–1315. responsible for the synthesis of tRNA (m1G) methyltransferase in Escherichia coli K-12. 2. Nureki O, et al. (2002) An enzyme with a deep trefoil knot for the active-site archi- Mol Gen Genet 188(3):440–446. tecture. Acta Crystallogr D Biol Crystallogr 58(Pt 7):1129–1137. 9. Björk GR, Wikström PM, Byström AS (1989) Prevention of translational frameshifting 3. Ahn HJ, et al. (2003) Crystal structure of tRNA(m1G37)methyltransferase: insights into by the modified nucleoside 1-methylguanosine. Science 244(4907):986–989. tRNA recognition. EMBO J 22(11):2593–2603. 10. Urbonavicius J, Qian Q, Durand JM, Hagervall TG, Björk GR (2001) Improvement of 4. Lim K, et al. (2003) Structure of the YibK methyltransferase from Haemophilus reading frame maintenance is a common function for several tRNA modifications. influenzae (HI0766): A cofactor bound at a site formed by a knot. Proteins 51(1): EMBO J 20(17):4863–4873. 56–67. 11. Persson BC, Bylund GO, Berg DE, Wikström PM (1995) Functional analysis of the ffh-trmD region 5. Elkins PA, et al. (2003) Insights into catalysis by a knotted TrmD tRNA methyltransferase. of the Escherichia coli chromosome by using reverse genetics. JBacteriol177(19):5554–5560. JMolBiol333(5):931–949. 12. Li J, Esberg B, Curran JF, Björk GR (1997) Three modified nucleosides present in the 6. Anantharaman V, Koonin EV, Aravind L (2002) SPOUT: A class of methyltransferases anticodon stem and loop influence the in vivo aa-tRNA selection in a tRNA-dependent that includes spoU and trmD RNA methylase superfamilies, and novel superfamilies of manner. J Mol Biol 271(2):209–221. predicted prokaryotic RNA methylases. J Mol Microbiol Biotechnol 4(1):71–75. 13. Thomas SR, Keller CA, Szyk A, Cannon JR, Laronde-Leblanc NA (2011) Struc- 7. Shao Z, et al. (2014) Crystal structure of tRNA m1G9 methyltransferase Trm10: Insight into the tural insight into the functional mechanism of Nep1/Emg1 N1-specific pseudouridine catalytic mechanism and recognition of tRNA substrate. Nucleic Acids Res 42(1):509–525. methyltransferase in ribosome biogenesis. Nucleic Acids Res 39(6):2445–2457.

8of9 | www.pnas.org/cgi/doi/10.1073/pnas.1422981112 Ito et al. Downloaded by guest on September 27, 2021 14. Wurm JP, et al. (2010) The ribosome assembly factor Nep1 responsible for Bowen- 30. Hill PJ, et al. (2013) Selective inhibitors of bacterial t-RNA-(N1G37) methyltransferase PNAS PLUS Conradi syndrome is a pseudouridine-N1-specific methyltransferase. Nucleic Acids Res (TrmD) that demonstrate novel ordering of the lid domain. J Med Chem 56(18): 38(7):2387–2398. 7278–7288. 15. Meyer B, et al. (2011) The Bowen-Conradi syndrome protein Nep1 (Emg1) has a dual 31. Christian T, Evilia C, Hou YM (2006) Catalysis by the second class of tRNA(m1G37) role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methyl transferase requires a conserved . Biochemistry 45(24):7463–7473. methylation of Ψ1191 in yeast 18S rRNA. Nucleic Acids Res 39(4):1526–1537. 32. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in os- 16. Christian T, Lahoud G, Liu C, Hou YM (2010) Control of catalytic cycle by a pair of cillation mode. Methods Enzymol 276:307–326. analogous tRNA modification enzymes. J Mol Biol 400(2):204–217. 33. Kabsch W (2010) XDS. Acta Crystallogr D Biol Crystallogr 66(Pt 2):125–132. 17. Christian T, Hou YM (2007) Distinct determinants of tRNA recognition by the TrmD 34. McCoy AJ, et al. (2007) Phaser crystallographic software. J Appl Cryst 40(Pt 4):658–674. and Trm5 methyl transferases. J Mol Biol 373(3):623–632. 35. Ito T, Yokoyama S (2010) Two enzymes bound to one transfer RNA assume alternative 18. Sakaguchi R, et al. (2012) Recognition of guanosine by dissimilar tRNA methyl- conformations for consecutive reactions. Nature 467(7315):612–616. – transferases. RNA 18(9):1687 1701. 36. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. 19. Sakaguchi R, Lahoud G, Christian T, Gamper H, Hou YM (2014) A divalent metal ion- Acta Crystallogr D Biol Crystallogr 66(Pt 4):486–501. 1 – dependent N -methyl transfer to G37-tRNA. Chem Biol 21(10):1351 1360. 37. Vagin AA, et al. (2004) REFMAC5 dictionary: Organization of prior chemical knowl- 20. Lahoud G, et al. (2011) Differentiating analogous tRNA methyltransferases by frag- edge and guidelines for its use. Acta Crystallogr D Biol Crystallogr 60(Pt 12 Pt 1): ments of the methyl donor. RNA 17(7):1236–1246. 2184–2195. 21. Craigen WJ, Cook RG, Tate WP, Caskey CT (1985) Bacterial peptide chain release 38. Brunger AT (2007) Version 1.2 of the crystallography and NMR system. Nat Protoc factors: Conserved primary structure and possible frameshift regulation of release 2(11):2728–2733. factor 2. Proc Natl Acad Sci USA 82(11):3616–3620. 39. Brünger AT, et al. (1998) Crystallography & NMR system: A new software suite for 22. Craigen WJ, Caskey CT (1986) Expression of peptide chain release factor 2 requires macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54(Pt 5): high-efficiency frameshift. Nature 322(6076):273–275. 905–921. 23. Enjalbert B, Letisse F, Portais JC (2013) Physiological and molecular timing of the 40. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macro- glucose to acetate transition in Escherichia coli. Metabolites 3(3):820–837. molecular structure solution. Acta Crystallogr D Biol Crystallogr 66(Pt 2):213–221. 24. Kao KC, Tran LM, Liao JC (2005) A global regulatory role of gluconeogenic genes in 41. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of Escherichia coli revealed by transcriptome network analysis. J Biol Chem 280(43): 36079–36087. progressive multiple sequence alignment through sequence weighting, position- – 25. Björk GR, et al. (2001) A primordial tRNA modification required for the evolution of specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22):4673 4680. life? EMBO J 20(1-2):231–239. 42. Gouet P, Courcelle E, Stuart DI, Métoz F (1999) ESPript: Analysis of multiple sequence – 26. Goto-Ito S, et al. (2008) Crystal structure of archaeal tRNA(m1G37)methyltransferase alignments in PostScript. Bioinformatics 15(4):305 308. aTrm5. Proteins 72(4):1274–1289. 43. Nureki O, et al. (2004) Deep knot structure for construction of active site and cofactor 27. Goto-Ito S, Ito T, Kuratani M, Bessho Y, Yokoyama S (2009) Tertiary structure checkpoint binding site of tRNA modification enzyme. Structure 12(4):593–602. at anticodon loop modification in tRNA functional maturation. Nat Struct Mol Biol 44. Leulliot N, Bohnsack MT, Graille M, Tollervey D, Van Tilbeurgh H (2008) The yeast 16(10):1109–1115. ribosome synthesis factor Emg1 is a novel member of the superfamily of alpha/beta 28. Christian T, Evilia C, Williams S, Hou YM (2004) Distinct origins of tRNA(m1G37) knot fold methyltransferases. Nucleic Acids Res 36(2):629–639.

methyltransferase. J Mol Biol 339(4):707–719. 45. Taylor AB, et al. (2008) The crystal structure of Nep1 reveals an extended SPOUT-class BIOCHEMISTRY 29. White TA, Kell DB (2004) Comparative genomic assessment of novel broad-spectrum methyltransferase fold and a pre-organized SAM-binding site. Nucleic Acids Res 36(5): targets for antibacterial drugs. Comp Funct Genomics 5(4):304–327. 1542–1554.

Ito et al. PNAS Early Edition | 9of9 Downloaded by guest on September 27, 2021